Biology subjects

Roy, J.

Publications and source records attributed to Roy, J..

At least 19 recordsLinked to original sources

bioRxiv2026

Gasdermin E couples mitochondrial stress to STING-driven neuronal pyroptosis during Chandipura virus encephalitis

Neurotropic RNA viruses are major causes of fatal encephalitis worldwide, yet how infected neurons transition from antiviral defense to inflammatory cell death is not well characterized. Chandipura virus (CHPV), an emerging neurotropic rhabdovirus, causes acute, rapidly progressive encephalitis with high case fatality in children, but the mechanisms underlying its neuropathogenesis remain poorly defined. Here, we demonstrate that CHPV suppresses canonical RNA virus sensing early but subsequently switches to a mitochondria-driven innate immune program that culminates in inflammatory cell death. Early infection of neuronal cells with CHPV was marked by reduced levels of the mitochondrial antiviral adaptor protein, MAVS and attenuation of type I and III interferon responses. As infection progressed, mitochondrial dysfunction promoted accumulation of mtROS, mitochondrial accumulation of cleaved GSDME and cytosolic mtDNA release, triggering STING activation, which coincided with robust neuroinflammation and pyroptotic cell death. Pharmacological inhibition or genetic silencing of STING markedly attenuated inflammatory signaling, prevented pyroptotic membrane rupture and protected neurons from cell death without significantly affecting viral replication. In contrast, GSDME depletion reduced both viral replication and neuronal death. Notably, GSDME depletion markedly attenuated STING phosphorylation, while STING depletion also reduced GSDME activation, revealing functional coupling between these pathways during CHPV-induced neuronal injury. Collectively, our findings identify a mitochondria-GSDME-STING axis linking early immune evasion to neuroinflammation during CHPV infection, revealing a previously unrecognized mechanism of inflammatory neuronal death in viral encephalitis and highlighting STING as a potential therapeutic target in certain CNS viral infections.

immunology↗

bioRxiv2026

The orphan receptor IL-17RD is a negative regulator of RIG-I-like receptor-dependent antiviral innate immunity and restrains SARS-CoV-2-induced lung inflammation

Detection of viral RNA by the RIG-I-like receptors (RLRs) RIG-I and MDA5 triggers assembly of a MAVS-dependent signalosome that activates the TBK1-IRF3 and IKK{beta}-NF-{kappa}B axes together with the JNK and p38 MAPK modules, driving type I and type III interferon (IFN) and inflammatory cytokine production. Because unrestrained activity of this pathway is a major cause of immunopathology, host-encoded negative regulators are essential, yet the full complement of these brakes remains incompletely defined. Here we identify interleukin-17 receptor D (IL-17RD, also known as SEF), an orphan member of the IL-17 receptor family previously characterized as an antagonist of FGF and Toll-like receptor signaling, as a negative regulator of RLR-driven antiviral innate immunity. Using a CRISPR-engineered and shRNA-depleted human airway epithelial-derived lung carcinoma A549 cell line, we show that loss of IL-17RD amplifies and prolongs phosphorylation of TBK1 and IRF3 in response to poly I:C transfection and to infection with encephalomyocarditis virus (EMCV) or Sendai virus (SeV), and likewise potentiates the IKK{beta}-I{kappa}B module and the TAK1-JNK1/2 and p38 MAPK branches. This translates into increased nuclear accumulation of IRF3 and p65, and markedly elevated induction of IFNB1, IFNL1-3, CCL5, IL6, and NFKBIA transcripts, as well as secreted IFN-{beta} and IL-6. Silencing IL-17RD in ACE2-expressing A549 cells similarly derepresses the antiviral and inflammatory transcriptional programme following SARS-CoV-2 infection. Epistasis experiments place IL-17RD at the level of MAVS, downstream of the RLR sentinels. Mechanistically, IL-17RD localizes to the ER-to-Golgi intermediate compartment (ERGIC), the membrane platform on which the MAVS signalosome is present, and associates with RIG-I, MDA5, MAVS, TBK1 and IRF3. Its re-expression in depleted cells redistributes RLR effectors and TRAF proteins across low-molecular-weight signalosome fractions, reducing the amount of IRF3 recruited to the 670 kDa MAVS signalosome complex. Complementation of IL-17RD-deficient cells also indicates that the intracellular TIR subdomain is sufficient to confer this antagonistic activity. Finally, Il17rd-/- mice display a splenic transcriptome enriched for antiviral response signatures, and, following intranasal infection with a moderate dose of SARS-CoV-2, they mount an exaggerated pulmonary cytokine response and develop significantly greater lung inflammation and fibrosis than wild-type littermates. Together, these data establish IL-17RD as a bona fide brake on the RLR-MAVS axis that limits virus-induced immunopathology, and identify the SEFIR/TIR subdomain as the module responsible for this activity.

microbiology↗

bioRxiv2026

Cross-species neural co-culture uncovers metabolic signatures of cellular crosstalk

Primates are distinguished by large brains relative to body size, with humans showing the greatest expansion. This increase in brain size evolved alongside advanced cognitive abilities as well as an elevated energetic demand. Importantly, allometric scaling alone does not explain this increased metabolic requirement, suggesting that other cellular mechanisms may be driving the unique energetic capacity of the human brain. Brain metabolism is critical for neurological function by providing the energy necessary for neuron firing. Much of metabolism in the brain is carried out by astrocytes: a type of glial cell that have long been viewed as passive support cells for neurons. More recent research has highlighted the unique roles of astrocytes in many critical neurological processes; however, it is less understood how astrocytes differ among species. To better characterize this, we developed a cross-species co-culture model of astrocytes and neurons from human or chimpanzee-derived iPSCs. This co-culture system allowed us to assess cell-type specific effects as well as species-specific differences in cellular interactions that may be driving overall differences in brain metabolism. We conducted single-cell RNA-sequencing as well as Seahorse XF Mitochondrial Stress tests and observed that human neural co-cultures are more metabolically active than chimpanzee neural co-cultures. Cross-species co-culture systems also highlight that astrocytes are driving major species differences in metabolism, whereas neurons are highly responsive to astrocytic activity. We conclude that both neurons and astrocytes have evolved differently across primates, and that metabolic interactions between these cell types are key contributors in primate brain evolution.

evolutionary biology↗

bioRxiv2026

Environmental filtering across seasons and host-associated selection shape the gut microbiome of sympatric European Sepsis dung flies

Animal microbiomes are shaped by both environmental exposure and host-associated filtering, but the relative importance of these processes remains poorly understood. Dung-associated insects provide an ideal model because they develop and feed in highly dynamic microbial environments. We investigated the gut microbiomes of six sympatric dung fly species of the genus Sepsis (Diptera: Sepsidae) and compared them with microbial communities in cow dung throughout a growing season in Switzerland. Using full-length 16S rRNA gene sequencing (PacBio), we characterized bacterial communities from 74 fly and 15 dung samples. Seasonal variation was the strongest predictor of microbiome composition, whereas host species exerted weaker effects that persisted after removing dung-associated taxa, indicating that gut communities are not merely passive reflections of environmental exposure. Only few gut microbiome reads were attributable to dung-associated taxa, and environmental overlap differed among fly species rather than season. A highly non-random core microbiome persisted across all six species: 36 bacterial genera (of 469) were shared by all hosts at [~]119-fold enrichment above random expectation and remained after removing dung-associated taxa. These findings support a two-layer model of microbiome assembly, in which seasonal environmental variation determines microbial availability while host-specific processes selectively retain a subset of taxa.

ecology↗

bioRxiv2026

A pistillate-biased epidermal regulatory program underlies glandular trichome development in Cannabis sativa

Glandular trichomes are specialized epidermal structures on Cannabis sativa L. inflorescences that synthesize and store cannabinoids and terpenoids, making them central to the species economic and medicinal value. Although trichome development is a multistage genetic process, its regulatory basis in C. sativa remains poorly understood, particularly with respect to sexual dimorphism and sex plasticity. Here, we combined orthologybased gene discovery with transcriptomic profiling to investigate trichome development across four floral phenotypes: female flowers (FF), male flowers (MF), induced male flowers on XX plants (IMF), and induced female flowers on XY plants (IFF). Using trichome development-related genes from Arabidopsis thaliana, a model for unicellular non-glandular trichome development, and Solanum lycopersicum, a model for multicellular glandular trichomes, we identified and mapped 53 candidate C. sativa trichome development regulator genes (CsTDRGs). The CsTDRG set did not support a simple Arabidopsis- or tomato-like model, but instead included Arabidopsis-like epidermal fate components, including MBW-related regulators and GL2, alongside tomato-like multicellular trichome regulators, including MIXTA-like, HD-ZIP IV, WOX, MTR, GRAS, and hormone-associated candidates. RNA-seq analysis showed that CsTDRG expression was more strongly associated with floral phenotype than chromosomal sex. Genes with preferential expression in pistillate tissues were CsTT8, CsMYC1/GL3, CsGL2, CsYABBY4 and CsMIXTA-like1/MYB106, whereas CsMTR1, CsGRAS9, and CsCKX3 were upregulated in male (MF and IMF) flower. These findings suggest that sexually dimorphic trichome development in C. sativa reflects differential regulation of a shared developmental toolkit that combines conserved epidermal fate components with multicellular and glandular trichome regulatory modules.

developmental biology↗

bioRxiv2026

Immune Biomarkers of Islet Transplant Rejection Revealed by Synthetic Immunological Niche

Islet transplantation can restore glycemic control in type 1 diabetes, yet the heterogeneity of patient immune responses and transplant outcomes motivates the need for technologies to monitor the graft. Since transplanted islets are not readily accessible for biopsy due to their diffuse engraftment within the liver, clinical monitoring relies on measurements such as islet mass, blood glucose, and C-peptide levels, which are lagging indicators that change only after substantial graft injury. Here, we developed a minimally invasive synthetic immunological niche (IN) that captures graft-associated immune responses through serial subcutaneous biopsy. We evaluated the IN across murine syngeneic, allogeneic, and autoimmune islet transplant models, including CD40/CD154 costimulatory blockade with anti-CD40L. In syngeneic versus allogeneic recipients, IN identified immune populations and transcriptomic signatures that mirrored the graft and distinguished healthy from rejecting grafts. In anti-CD40L treated allografts, IN revealed innate macrophage- and dendritic cell-associated programs linked to graft acceptance versus rejection, whereas IN from untreated allografts showed stronger adaptive immune signatures. Longitudinal IN profiling further detected progressive inflammatory activation in accepted allografts, indicating persistent subclinical risk. Finally, in an autoimmune allograft model treated with anti-CD40L plus rapamycin, IN identified a 13-gene signature that separated early from late rejection trajectories and distinguished autoimmune-from alloimmune-associated rejection programs. Overall, these findings establish IN as a surrogate tissue for minimally invasive monitoring of islet graft and early detection of rejection-associated immune dysregulation. One Sentence SummaryAn engineered immunological niche captures distinct immune signatures of allo- and auto-mediated islet transplant rejection

bioengineering↗

bioRxiv2026

Ethylene-Gibberellin Crosstalk Drives Phenotypic Sex Changes in Cannabis sativa

Abstract/SummarySex expression in Cannabis sativa is determined by XX/XY sex chromosomes but remains plastic, with ethylene inhibition inducing male flowers on XX plants and ethylene release inducing female flowers on XY plants. Although ethylene is a central regulator of this process, the contribution of gibberellin signaling to cannabis sex reversal remains poorly defined. Here, we reconstructed the GA biosynthesis, regulation, and signaling pathway in C. sativa and profiled GA-related gene expression during chemically induced sex reversal. Orthology-based searches identified 50 putative C. sativa GA-related genes, widely distributed across the genome, with the X chromosome harboring 11 genes, including six within the non-recombining region. Transcriptomic analyses across vegetative baseline, early post-treatment leaves, and developing flowers showed that expression profiles were broadly similar between XX and XY plants at day 0, weakly perturbed at day 1, and strongly structured by floral phenotype at day 14. Early responses were limited to downregulation of CsGA3ox1 in ethephon-treated XY plants and CsGASA1 in STS-treated XX plants. By day 14, sex reversal was associated with differential expression of key genes, including CsGA1, multiple GA20ox orthologs, CsGID1B, CsSLY2, and several GASA genes, indicating broad remodeling of GA regulation. Our findings position the GA pathway as a downstream module of ethylene-driven sex reversal in C. sativa, with GA activity tracking floral sexual identity, extending the framework of sexual plasticity beyond ethylene, and identifying candidate genes for functional validation and the development of sex-stable cultivars.

developmental biology↗

bioRxiv2026

Synthetic Immunological Niche Reveals Early Immune Dysregulationand Stratifies Therapeutic Response in Type 1 Diabetes

In Type 1 Diabetes (T1D), disease onset and response to immunotherapy vary widely among individuals, reflecting heterogeneous stage-specific immune dysregulation that remains undefined. To investigate this heterogeneity, we engineered a microporous polycaprolactone scaffold that forms a synthetic immunological niche (IN) upon subcutaneous implantation, enabling in vivo capture of systemic immune dysregulation. In non-obese diabetic (NOD) mice, longitudinal transcriptomic profiling of IN-infiltrating cells identified early-stage genes relatively enriched for myeloid cells, followed by progressive increases in T cell-associated dysregulation at later stages that distinguished T1D progressors from non-progressors. We derived an early-stage IN-based T1D gene signature capturing immune alterations. The signature stratified NOD progressors from non-progressors as early as 6 weeks of age and was conserved across human T1D datasets, distinguishing T1D from non-diabetic individuals in spleen and pancreatic lymph node samples, but not peripheral blood. Signature-based stratification further revealed enrichment of macrophage-associated TNF- pathways in NOD progressors, validated in human T1D islets. Given heterogeneous response to anti-TNF- therapy, IN profiling identified resistance-associated mechanisms and enabled derivation of a pathway score that prospectively distinguished treatment-sensitive from resistant mice prior to therapy, establishing the IN as a minimally invasive platform for detecting stage-wise immune dysregulation and stratifying immunotherapy response in T1D.

bioengineering↗

bioRxiv2026

A Drosophila screen identifies domino as a link between chromatin regulation and synaptic organization

While heterozygous gene-disrupting variants in dosage-sensitive genes are strongly implicated in autism spectrum disorder (ASD), their effects on behavior in vivo remain poorly understood. To address this, we conducted a targeted behavioral screen in Drosophila using high-confidence ASD risk genes. This screen identified 48 lines with altered sleep, activity, or social behavior, including many genes not previously known to regulate these behaviors. The chromatin remodeler domino (dom) emerged as a compelling hit. Heterozygous mutants showed altered social spacing and male-biased changes in sleep and activity. RNA-sequencing revealed changes in gene expression and splicing associated with synaptic pathways. Consistent with these molecular changes, immunofluorescence revealed increased presynaptic activity in a brain region associated with sleep and sensory processing. Together, these findings show that partial loss of ASD risk genes is sufficient to alter behavior and identify dom as a link between transcriptional regulation, synaptic organization and behavior.

neuroscience↗

bioRxiv2025

Indoor Rewilding of Laboratory Mice Recalibrates Pulmonary Mucosal Immunity and Mechanics

Laboratory mice raised under specific-pathogen-free (SPF) conditions experience restricted microbial and antigenic exposure, which favours an immature immune system and limits their translational value for respiratory research. While microbial enrichment in "dirty" mouse models restores immune maturation, its impact on integrated respiratory function and model transferability to human disease remains understudied. Here, we tested whether ecological exposure through indoor rewilding of SPF-reared mice could reshape immune complexity and recalibrate pulmonary physiology. Two-month-old female C57BL/6J mice were housed for three months under SPF or indoor-rewilding conditions and assessed for immune, mechanical, and systemic parameters. Rewilded mice exhibited expanded pulmonary immune subsets, increased dendritic-cell immune checkpoint, with TNF/IFN-{gamma} activation coupled to regulatory IL-10 signaling. Despite sustained exposure, the alveolar-capillary barrier integrity was preserved. Functionally, respiratory oscillometry revealed improved pulmonary mechanics, including lower airway resistance, higher compliance, and reduced airway responsiveness to methacholine. Systemic cytokine analyses indicated compartmentalized pulmonary immune activation, maintaining an overall anti-inflammatory balance. Importantly, PRIA screening detected no reportable pathogens introduced during rewilding, while cecal shotgun metagenomics confirmed microbial enrichment. Together, these findings demonstrate that indoor rewilding reestablishes coordinated lung immune and mechanical homeostasis in SPF-reared mice, providing a safe and scalable model for studying human-like mucosal immunity and respiratory physiology with broad implications for preclinical respiratory research and therapeutic testing.

immunology↗

bioRxiv2025

Similar destabilization of neural dynamics under different general anesthetics

Different classes of anesthetics can induce unconsciousness despite acting through distinct biological mechanisms. This raises the possibility that they produce a convergent effect on the dynamics or temporal evolution of neural population activity. To explore this, we analyzed intracortical electrophysiological recordings during infusions of propofol, ketamine, and dexmedetomidine, using a rigorous method to estimate dynamical stability. We found that all three anesthetics, despite their molecular differences, similarly affect cortical states by destabilizing their dynamics. This destabilization matched the slower recovery from sensory perturbations and longer stimulus-induced autocorrelation times observed during the anesthetic infusions. The destabilization was also reflected predominantly in lower-frequency ranges, linking it to the well-known increase in low-frequency power during anesthesia. Finally, destabilization closely tracked real-time fluctuations in consciousness. Together, these findings suggest that cortical destabilization may be a shared neural correlate of anesthetic-induced unconsciousness, offering a mechanistic explanation for low-frequency oscillations observed during anesthesia.

neuroscience↗

bioRxiv2025

Characterization of interindividual DNA methylation variability in rainbow trout (Oncorhynchus mykiss)

Interindividual epigenetic variability, particularly in DNA methylation, is now recognized as a significant contributor to phenotypic diversity in humans and mammals. These epivariable regions, which make up a small fraction of the genome, are strongly influenced by genetic factors and environmental factors, especially during early development. In this context, epigenetic variability of DNA methylation has been proposed as an adaptive force involved in various environmental responses. In fish and other vertebrates, environmental factors are known to influence the health, performance and welfare, likely through the alteration of the epigenetic landscape. However, whether interindividual epigenetic variability may contribute to the phenotypic plasticity of fishes is unknown. Here we provide a first description of the rainbow trout methylome variability using a whole-genome bisulfite sequencing approach in an isogenic line to minimize genetic variation. Variable methylation regions were identified in both liver and hypothalamus tissues of 12 replicate fishes and were found enriched at gene regulatory elements, such as promoters and first introns. Gene Ontology analysis revealed functional clusters related to cellular development, neural communication, metabolic balance, and immune response. Interestingly, some variably methylated regions are found at the same genomic loci in both tissues and showed a strong intraindividual correlation in methylation levels, suggesting establishment during early embryogenesis. Overall, our work demonstrates the existence of interindividual epigenetic variability in rainbow trout and provides valuable insights into the regulatory function of DNA methylation variation that is likely involved in developmental and physiological processes.

genomics↗

bioRxiv2025

Boltz-2: Towards Accurate and Efficient Binding Affinity Prediction

Accurately modeling biomolecular interactions is a central challenge in modern biology. While recent advances, such as AlphaFold3 and Boltz-1, have substantially improved our ability to predict biomolecular complex structures, these models still fall short in predicting binding affinity, a critical property underlying molecular function and therapeutic efficacy. Here, we present Boltz-2, a new structural biology foundation model that exhibits strong performance for both structure and affinity prediction. Boltz-2 introduces controllability features including experimental method conditioning, distance constraints, and multi-chain template integration for structure prediction, and is, to our knowledge, the first AI model to approach the performance of free-energy perturbation (FEP) methods in estimating small molecule-protein binding affinity. Crucially, it achieves strong correlation with experimental readouts on many benchmarks, while being at least 1000x more computationally efficient than FEP. By coupling Boltz-2 with a generative model for small molecules, we demonstrate an effective workflow to find diverse, synthesizable, high-affinity binders, as estimated by absolute FEP simulations on the TYK2 target. To foster broad adoption and further innovation at the intersection of machine learning and biology, we are releasing Boltz-2 weights, inference, and training code 1 under a permissive open license, providing a robust and extensible foundation for both academic and industrial research.

molecular biology↗

bioRxiv2025

Sex-Specific Ethylene Responses and Convergent Plasticity: Multi-Omic Insights into Cannabis Sexual Plasticity

Cannabis sativa L. exhibits remarkable sexual plasticity: both XX and XY individuals can undergo complete phenotypic sex reversal in response to ethylene modulation. While this phenomenon is well documented, the molecular mechanisms remain underexplored. Here, we present the first multi-omic study of hormonally induced sex change in both XX and XY Cannabis plants, integrating transcriptomic profiling, ethylene pathway metabolite quantification, and whole-genome sequencing across three genetically distinct genotypes. Treatments with silver thiosulfate (STS) and ethephon induced >80% phenotypic conversion, but transcriptomic responses diverged sharply between chromosomal sexes. We profiled 47 ethylene-related genes (ERGs) and identified 14 high-confidence candidates--including CsACS1, CsACO5, CsERF1, and CsMTN--with sex-specific, time-dependent expression patterns that support a two-phase model of plasticity: early transcriptional reprogramming followed by stabilization of new floral identities. Several candidate ERGs were in non-recombining regions of the X chromosome or absent from the Y, while most showed low nucleotide diversity, suggesting functional constraint. These findings provide a high-resolution view of ethylene-responsive sex plasticity and demonstrate that convergent floral phenotypes arise from distinct regulatory programs in XX and XY plants. Our work advances the molecular understanding of sexual plasticity in dioecious species and identifies candidate genes for the development of sex-stable cultivars in Cannabis and other crops.

developmental biology↗

bioRxiv2025

Male gut microbiome mediates post-mating sexual selection in Drosophila

When females mate with multiple males, sexual selection continues after mating and favors males with more competitive ejaculates. While these traits are physiologically costly and sensitive to internal state, the effects of the gut microbiome on male reproductive performance remains underexplored. Here, we tested how gut microbial communities influence sperm competition outcomes and the traits underpinning them by inoculating male Drosophila melanogaster with fully factorial synthetic consortia of five common commensal bacteria. We found highly context-dependent and non-additive microbial effects, along with two general patterns: Acetobacter-dominated microbiomes enhanced sperm transfer and increased paternity shares by 20-27% relative to germ-free males, whereas species richness strongly predicted sperm viability and female sperm storage. These results indicate that gut microbes contribute to multiple components of ejaculate performance through distinct pathways, potentially including nutrient provisioning for spermatogenesis, acetate-mediated energy metabolism, and microbe-dependent modulation of female sperm use. By integrating microbial ecology with reproductive biology, our findings establish the gut microbiome as an overlooked but fundamental modulator of sexually selected traits. This perspective helps clarify how non-genetic and community-based factors generate phenotypic variation under strong selection and provides a framework for future work on the mechanisms, transmission routes, and evolutionary consequences of host-microbe interactions in reproduction.

evolutionary biology↗

bioRxiv2025

A domestication change at PvMYB26 in common bean sheds light on the origins of Middle American agriculture

Domestication imposed radically different selection pressures on plants, eventually transforming them into the cultivated forms that support global populations today. Here, we investigate the loss of seed dispersal via pod shattering during common bean (Phaseolus vulgaris L.) domestication. We identified an eight-kb deletion eliminating the transcription start site and promoter of the candidate gene PvMYB26. Mutants express PvMYB26 at < 1% of the level of wild types and produce 44% less pod lignin. Whole-genome sequencing revealed that the mutation is nearly diagnostic for domestication status among Middle American common bean, indicating its importance in domestication. We also identified a high-frequency PvMYB26 frameshift/premature stop mutation unique to Andean domesticates. Wild haplotypes most like Middle American domesticates are found in eastern Jalisco, Mexico. Our results suggest that West-Central Mexico was the site of common bean domestication and suggest that this region may have been important in the rise of Middle American agriculture.

evolutionary biology↗

bioRxiv2025

DESeq2-MultiBatch: Batch Correction for Multi-Factorial RNA-seq Experiments

RNA sequencing (RNA-seq) experiments frequently encounter batch effects that can significantly distort biological interpretations, particularly in complex, multifactorial studies where biological variables interact with experimental batch conditions. Existing batch correction tools primarily address technical variability and often neglect these critical interaction effects, resulting in incomplete adjustments. To address this gap, we introduce DESeq2-MultiBatch, a novel, lightweight batch correction method implemented entirely within the DESeq2 analytical framework. Unlike conventional approaches, DESeq2-MultiBatch directly leverages DESeq2s internal model estimates to correct raw gene count data, accurately adjusting for experimental batch effects, including interactions with biological variables. Here, we demonstrate that DESeq2-MultiBatch effectively mitigates batch-related variability while preserving genuine biological differences. Benchmarking against widely used methods highlights DESeq2-MultiBatch as a robust, practical solution for improving exploratory data visualization and downstream analyses in multifactorial RNA-seq studies. Availability and implementationhttps://github.com/julienroyulaval/DESeq2-MultiBatch Contactjulien.roy.10@ulaval.ca Supplementary informationSupplementary data are available at Bioinformatics online.

bioinformatics↗

bioRxiv2025

Olfactory receptor 2 drives abdominal aortic aneurysm by promoting CX3CR1-mediated monocyte recruitment

BackgroundAbdominal aortic aneurysms (AAA) are characterized by an intricate interplay of extracellular matrix degradation and inflammation. Macrophages are centrally involved in these processes. The mechanisms underlying macrophage activation in AAA remain incompletely understood. Vascular macrophages have been shown to express olfactory receptor 2 (Olfr2), a G-protein coupled receptor involved in mediating the sense of smelling and regulating inflammatory activity in macrophages. Whether Olfr2 plays a role in modulating macrophage responses in AAA formation remains unknown. Methods & ResultsIn silico micro-array analysis showed increased expression of the human Olfr2 orthologue OR6A2 in AAA tissue compared to healthy aorta. Flow cytometric analysis revealed increased expression of OR6A2 on classical, non-classical, and intermediate monocytes of patients with a large AAA (> 5cm) in comparison to patients with smaller AAA (< 5cm). Up to 30% of vascular macrophages expressed OR6A2 in human AAA and Olfr2 in mouse AAA tissue. Olfr2 expression peaked on major histocompatibility complex II-high (MHCIIhigh) and C-C chemokine receptor type 2-low (CCR2low) aortic monocytes and macrophages on day 7 following experimental AAA initiation and decreased to baseline expression on day 28. Olfr2 gene (Olfr2-/-) deficiency protected mice from AAA formation, which was accompanied by lowered ECM degradation, reduced macrophage infiltration and increased smooth muscle cell content. Conversely, treatment with the Olfr2 agonist octanal exacerbated AAA formation and inflammation, while the antagonist citral reduced AAA formation in comparison to vehicle treated mice. Bulk transcriptome analysis of aortic tissue revealed reduced inflammatory gene expression in Olfr2-/- mice at day 7 following AAA initiation. Spectral flow cytometry resolved 20 aortic immune cell populations, which were largely reduced in quantity by Olfr2-deficiency at day 7 and day 28 post experimental AAA formation, while circulating leukocyte counts and monocyte subset distribution were not altered between Olfr2+/+ and Olfr2-/- mice. Circulating Ly6Chigh-monocytes exhibited reduced expression of the CX3C motif chemokine receptor 1 (CX3CR1) and CCR2 during AAA formation. Transcriptional analysis of monocytes confirmed downregulation of pathways associated with cell adhesion, motility and migration. In vitro, Olfr2-/- monocytes showed impaired migration towards the CX3CR1 ligand CX3CL1. Competitive transfer of Olfr2+/+ and Olfr2-/- monocytes confirmed reduced migratory capacity of Olfr2-/- monocytes into the developing AAA. ConclusionWe demonstrate a critical relevance for Olfr2 in the modulation of the inflammatory response underlying AAA, which is mediated by enhanced monocyte recruitment.

immunology↗